Jimenez, Fernanda2020-08-252020-08-252018-05https://hdl.handle.net/11299/215190University of Minnesota Ph.D. dissertation. May 2018. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Daniel Bond. 1 computer file (PDF); 151 pages.Microbial metabolism represents a rich source of catalytic tools available for biotechnological applications. Microbial metal reduction is no exception, as it represents a mechanism for transferring electrons stored within nonreactive organic compounds to inorganic redox-active compounds. Despite the usefulness of organisms capable of metal reduction, not enough is known about their electron transfer pathway to be able to engineer them for real-world applications. Specifically, how electrons cross cell membranes to be available on the extracellular surface where they can react with metals or electrodes remains one of the least well-studied aspects of extracellular electron transfer. In this thesis, a compilation of studies into extracellular electron transfer pathways of the model organism Geobacter sulfurreducens, and the characteristics of current-producing biofilms produced by this species uncovers fundamental steps and bottlenecks in this metabolic strategy. Through isotopic label incorporation, the first spatially resolved direct measurement of anabolic activity in G. sulfurreducens biofilms was obtained, concluding that metabolic activity is constrained to the layers within the first few microns from the electrode surface. Combinatorial deletion of putative outer membrane electron conduit gene clusters followed by analysis of the ability of this mutant collection to reduce different extracellular substrates showed that several electron conduit gene clusters are necessary in tandem during metal reduction, while only a previously unstudied gene cluster extABCD was necessary for wild type levels of electrode reduction. Within this mutant collection, the strain lacking all studied gene clusters except extABCD (extABCD+), reached higher current densities with faster doubling times than wild type. The extABCD+ strain was found to form biofilms containing 30% more cells than wild type at the electrode:biofilm interface, where isotopic label incorporation showed 38% higher anabolic activity per cell in extABCD+ biofilms compared to wild type. Thus, by focusing on the fundamental physiology of extracellular electron transfer, evidence for substrate-specific electron transfer pathways were found, and a strain with a streamlined pathway for increased electrode reduction could be constructed. The findings in this work suggest a route to engineering organisms with metal- or electrode-specific reduction pathways, and enhancing electron transfer rates in these systems.enelectron conduitGeobactermultiheme cytochromeThesis or Dissertation